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. 2009 Jun 3;83(16):8233–8246. doi: 10.1128/JVI.02672-08

FIG. 7.

FIG. 7.

Influenza A virus replication is dependent upon opposite virus-induced effects on Bax and Bak activity that are unlikely to be interferon related. (A) Influenza A virus replication was analyzed by plaque assay. Virus replication is severely attenuated in Bax KO cells, resulting in a 2-log decrease in PFU/ml compared to the WT. Bak KO cells allow a maximum replication similar to that of the WT, while Bax/Bak DKO cells show a slight elevation of infectious titers during infection. These results indicate that Bax is proviral during infection, while Bak is dispensable for replication. (B) Bax was transiently expressed in all cell types by Lipofectamine 2000 transfection of a C2-Bax-GFP construct prior to infection, and supernatant samples were collected for plaque assay at 48 hpi. Baseline virus replication in each cell type was evaluated using empty C2-GFP plasmid transfection. Bax reconstitution in Bax KO cells resulted in a fivefold increase in infectious titers compared to the control (P = 0.0007). A minimal effect on the virus titer was seen after Bax overexpression by transient transfection in WT cells compared to empty plasmid controls. (C) Influenza A virus replication was assessed by reverse transcription-PCR. Serial dilutions of stock virus at known concentrations were also analyzed to generate a standard curve to which experimental samples were compared, thus calculating the approximate number of influenza A virus particles/ml in each sample. By 24 hpi, Bax KO, Bak KO, and Bax/Bak DKO cells all showed significantly higher levels of influenza A virus RNA released into the cell culture supernatant than did WT cells. (D) Interferon activity was assessed by infecting mock- and influenza A virus-infected cells with interferon-sensitive, GFP-linked NDV and quantifying the mean GFP expression levels of 10,000 events per condition by FACS analysis. Each assay was run in triplicate, and data are expressed as the ratio of the numbers of influenza A virus-infected to mock-infected cells per cell type. After influenza A virus infection, Bak KO cells exhibited a slight decrease in ratio compared to the WT, representing a 30% increase in interferon activity (P = 0.002). Bax KO and Bax/Bak DKO cells both showed similar fluorescence changes compared to the WT after infection. Due the high degree of similarity between cell types, these results suggest that the interferon response in infected cells is modulated by viral replication in the presence of Bak and is only slightly modified by Bax activity during influenza A virus infection. As an elevated interferon response typically leads to a reduced virus replication capacity, these results also suggest that it is unlikely that the observed trends in infectious virus titer are due to virus-induced interferon signaling.